Differential regulation of glyceroneogenesis by glucocorticoids in epididymal and retroperitoneal white adipose tissue from rats.

PURPOSE: Investigate the glycerol-3-phosphate generation pathways in epididymal (EPI) and retroperitoneal (RETRO) adipose tissues from dexamethasone-treated rats. METHODS: Rats were treated with dexamethasone for 7 days. Glycerol-3-phosphate generation pathways via glycolysis, glyceroneogenesis and direct phosphorylation of glycerol were evaluated, respectively, by 2-deoxyglucose uptake, phosphoenolpyruvate carboxykinase (PEPCK-C) activity and pyruvate incorporation into triacylglycerol (TAG)-glycerol, and glycerokinase activity and glycerol incorporation into TAG-glycerol. RESULTS: Dexamethasone treatment markedly decreased the body weight, but increased the weight and lipid content of EPI and RETRO and plasma insulin, glucose, non-esterified fatty acid and TAG levels. EPI and RETRO from dexamethasone-treated rats showed increased rates of de novo fatty acid synthesis (80 and 100%) and basal lipolysis (20%). In EPI, dexamethasone decreased the 2-deoxyglucose uptake (50%), as well as glyceroneogenesis, evidenced by a decrease of PEPCK-C activity (39%) and TAG-glycerol synthesis from pyruvate (66%), but increased the glycerokinase activity (50%) and TAG-glycerol synthesis from glycerol (72%) in this tissue. In spite of a similar reduction in 2-deoxyglucose uptake in RETRO, dexamethasone treatment increased glyceroneogenesis, evidenced by PEPCK activity (96%), and TAG-glycerol synthesis from pyruvate (110%), accompanied by a decrease in glycerokinase activity (50%) and TAG-glycerol synthesis from glycerol (50%). Dexamethasone effects on RETRO were accompanied by a decrease in p-Akt content and by lower insulin effects on the rates of glycerol release in the presence of isoproterenol and on the rates of glucose uptake in isolated adipocytes. CONCLUSION: Our data demonstrated differential regulation of glyceroneogenesis and direct phosphorylation of glycerol by glucocorticoids in EPI and RETRO from rats.

In vitro TNF-α- and noradrenaline-stimulated lipolysis is impaired in adipocytes from growing rats fed a low-protein, high-carbohydrate diet.

The aim of this study was to investigate tumor necrosis factor alpha (TNF-α)- and noradrenaline (NE)-stimulated lipolysis in retroperitoneal (RWAT) and epididymal (EAT) white adipose tissue as a means of understanding how low-protein, high-carbohydrate (LPHC) diet-fed rats maintain their lipid storage in a catabolic environment (marked by increases in serum TNF-α and corticosterone and sympathetic flux to RWAT and EAT), as previously observed. Adipocytes or tissues from the RWAT and EAT of rats fed an LPHC diet and rats fed a control (C) diet for 15 days were used in the experiments. The adipocytes from both tissues of the LPHC rats exhibited lower TNF-α- stimulated lipolysis compared to adipocytes from the C rats. The intracellular lipolytic agents IBMX, DBcAMPc and FSK increased lipolysis in both tissues from rats fed the C and LPHC diets compared to basal lipolysis; however, the effect was approximately 2.5-fold lower in adipocytes from LPHC rats. The LPHC diet induced a marked reduction in the ß3 and α2-AR, adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) content in RWAT and EAT. The LPHC diet did not affect TNF-α receptor 1 content but did induce a reduction in ERK p44/42 in both tissues. The present work indicates that RWAT and EAT from LPHC rats have an impairment in the lipolysis signaling pathway activated by NE and TNF-α, and this impairment explains the reduced response to these lipolytic stimuli, which may be fundamental to the maintenance of lipid storage in LPHC rats.

Hydrogen peroxide production regulates the mitochondrial function in insulin resistant muscle cells: effect of catalase overexpression.

The mitochondrial redox state plays a central role in the link between mitochondrial overloading and insulin resistance. However, the mechanism by which the ROS induce insulin resistance in skeletal muscle cells is not completely understood. We examined the association between mitochondrial function and H2O2 production in insulin resistant cells. Our hypothesis is that the low mitochondrial oxygen consumption leads to elevated ROS production by a mechanism associated with reduced PGC1α transcription and low content of phosphorylated CREB. The cells were transfected with either the encoded sequence for catalase overexpression or the specific siRNA for catalase inhibition. After transfection, myotubes were incubated with palmitic acid (500µM) and the insulin response, as well as mitochondrial function and fatty acid metabolism, was determined. The low mitochondrial oxygen consumption led to elevated ROS production by a mechanism associated with ß-oxidation of fatty acids. Rotenone was observed to reduce the ratio of ROS production. The elevated H2O2 production markedly decreased the PGC1α transcription, an effect that was accompanied by a reduced phosphorylation of Akt and CREB. The catalase transfection prevented the reduction in the phosphorylated level of Akt and upregulated the levels of phosphorylated CREB. The mitochondrial function was elevated and H2O2 production reduced, thus increasing the insulin sensitivity. The catalase overexpression improved mitochondrial respiration protecting the cells from fatty acid-induced, insulin resistance. This effect indicates that control of hydrogen peroxide production regulates the mitochondrial respiration preventing the insulin resistance in skeletal muscle cells by a mechanism associated with CREB phosphorylation and ß-oxidation of fatty acids.

Increased glyceride-glycerol synthesis in liver and brown adipose tissue of rat: in-vivo contribution of glycolysis and glyceroneogenesis.

We have previously shown that a high-protein, carbohydrate-free diet can decrease the production of glycerol-3-phosphate (G3P) from glucose and increase glyceroneogenesis in both brown (BAT) and epididymal (EAT) adipose tissue. Here, we utilized an in-vivo approach to examine the hypothesis that there is reciprocal regulation in the G3P synthesis from glucose (via glycolysis) and glyceroneogenesis in BAT, EAT and liver of fasted rats and cafeteria diet-fed rats. Glyceroneogenesis played a prominent role in the generation of G3P in the liver (~70 %) as well as in BAT and EAT (~80 %) in controls rats. The cafeteria diet induced an increase in the total glyceride-glycerol synthesis and G3P synthesis from glucose and a decrease in glyceroneogenesis in BAT; this diet did not affect either the total glyceride-glycerol synthesis or G3P generation from glyceroneogenesis or glycolysis in the liver or EAT. Fasting induced an increase in total glyceride-glycerol synthesis and glyceroneogenesis and a decrease in G3P synthesis from glucose in the liver but did not affect either the total glyceride-glycerol synthesis or G3P synthesis from glyceroneogenesis in BAT and EAT, despite a reduction in glycolysis in these tissues. These data demonstrate that reciprocal changes in the G3P generation from glucose and from glyceroneogenesis in the rat liver and BAT occur only when the synthesis of glycerides-glycerol is increased. Further, our data suggest that this increase may be essential for the systemic recycling of fatty acids by the liver from fasted rats and for the maintenance of the thermogenic capacity of BAT from cafeteria diet-fed rats.

The sympathetic nervous system regulates the three glycerol-3P generation pathways in white adipose tissue of fasted, diabetic and high-protein diet-fed rats.

The aim of the present study was to investigate the participation of the sympathetic nervous system (SNS) in the control of glycerol-3-P (G3P) generating pathways in white adipose tissue (WAT) of rats in three situations in which the plasma insulin levels are low. WAT from 48 h fasted animals, 3 day-streptozotocin diabetic animals and high-protein, carbohydrate-free (HP) diet-fed rats was surgical denervated and the G3P generation pathways were evaluated. Food deprivation, diabetes and the HP diet provoke a marked decrease in the rate of glucose uptake and glycerokinase (GyK) activity, but a significant increase in the glyceroneogenesis, estimated by the phosphoenolpyruvate carboxykinase (PEPCK) activity and the incorporation of 1-[(14)C]-pyruvate into glycerol-TAG. The denervation provokes a reduction (~70%) in the NE content of WAT in fasted, diabetic and HP diet-fed rats. The denervation induced an increase in WAT glucose uptake of fed, fasted, diabetic and HP diet-fed rats (40%, 60%, 3.2 fold and 35%, respectively). TAG-glycerol synthesis from pyruvate was reduced by denervation in adipocytes of fed (58%) and fasted (36%), saline-treated (58%) and diabetic (23%), and HP diet-fed rats (11%). In these same groups the denervation reduced the PEPCK mRNA expression (75%-95%) and the PEPCK activity (35%-60%). The denervation caused a ~35% decrease in GyK activity of control rats and a further ~35% reduction in the already low enzyme activity of fasted, diabetic and HP diet-fed rats. These data suggest that the SNS plays an important role in modulating G3P generating pathways in WAT, in situations where insulin levels are low.

A low-protein, high-carbohydrate diet increases fatty acid uptake and reduces norepinephrine-induced lipolysis in rat retroperitoneal white adipose tissue.

A low-protein, high-carbohydrate (LPHC) diet for 15 days increased the lipid content in the carcass and adipose tissues of rats. The aim of this work was to investigate the mechanisms of this lipid increase in the retroperitoneal white adipose tissue (RWAT) of these animals. The LPHC diet induced an approximately two- and tenfold increase in serum corticosterone and TNF-α, respectively. The rate of de novo fatty acid (FA) synthesis in vivo was reduced (50%) in LPHC rats, and the lipoprotein lipase activity increased (100%). In addition, glycerokinase activity increased (60%), and the phosphoenolpyruvate carboxykinase content decreased (27%). Basal [U-¹4C]-glucose incorporation into glycerol-triacylglycerol did not differ between the groups; however, in the presence of insulin, [U-¹4C]-glucose incorporation increased by 124% in adipocytes from only control rats. The reductions in IRS1 and AKT content as well as AKT phosphorylation in the RWAT from LPHC rats and the absence of an insulin response suggest that these adipocytes have reduced insulin sensitivity. The increase in NE turnover by 45% and the lack of a lipolytic response to NE in adipocytes from LPHC rats imply catecholamine resistance. The data reveal that the increase in fat storage in the RWAT of LPHC rats results from an increase in FA uptake from circulating lipoproteins and glycerol phosphorylation, which is accompanied by an impaired lipolysis that is activated by NE.

Several agents and pathways regulate lipolysis in adipocytes.

Adipose tissue is the only tissue capable of hydrolyzing its stores of triacylglycerol (TAG) and of mobilizing fatty acids and glycerol in the bloodstream so that they can be used by other tissues. The full hydrolysis of TAG depends on the activity of three enzymes, adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and monoacylglycerol lipase, each of which possesses a distinct regulatory mechanism. Although more is known about HSL than about the other two enzymes, it has recently been shown that HLS and ATGL can be activated simultaneously, such that the mechanism that enables HSL to access the surface of lipid droplets also permits the stimulation of ATGL. The classical pathway of lipolysis activation in adipocytes is cAMP-dependent. The production of cAMP is modulated by G-protein-coupled receptors of the Gs/Gi family and cAMP degradation is regulated by phosphodiesterase. However, other pathways that activate TAG hydrolysis are currently under investigation. Lipolysis can also be started by G-protein-coupled receptors of the Gq family, through molecular mechanisms that involve phospholipase C, calmodulin and protein kinase C. There is also evidence that increased lipolytic activity in adipocytes occurs after stimulation of the mitogen-activated protein kinase pathway or after cGMP accumulation and activation of protein kinase G. Several agents contribute to the control of lipolysis in adipocytes by modulating the activity of HSL and ATGL. In this review, we have summarized the signalling pathways activated by several agents involved in the regulation of TAG hydrolysis in adipocytes.

Adipose overexpression of desnutrin promotes fatty acid use and attenuates diet-induced obesity.

OBJECTIVE: To investigate the role of desnutrin in adipose tissue triacylglycerol (TAG) and fatty acid metabolism. RESEARCH DESIGN AND METHODS: We generated transgenic mice overexpressing desnutrin (also called adipose triglyceride lipase [ATGL]) in adipocytes (aP2-desnutrin) and also performed adenoviral-mediated overexpression of desnutrin in 3T3-L1CARDelta1 adipocytes. RESULTS: aP2-desnutrin mice were leaner with decreased adipose tissue TAG content and smaller adipocyte size. Overexpression of desnutrin increased lipolysis but did not result in increased serum nonesterified fatty acid levels or ectopic TAG storage. We found increased cycling between diacylglycerol (DAG) and TAG and increased fatty acid oxidation in adipocytes from these mice, as well as improved insulin sensitivity. CONCLUSIONS: We show that by increasing lipolysis, desnutrin overexpression causes reduced adipocyte TAG content and attenuation of diet-induced obesity. Desnutrin-mediated lipolysis promotes fatty acid oxidation and re-esterification within adipocytes.

Fatty acid synthesis and generation of glycerol-3-phosphate in brown adipose tissue from rats fed a cafeteria diet.

In vivo fatty acid synthesis and the pathways of glycerol-3-phosphate (G3P) production were investigated in brown adipose tissue (BAT) from rats fed a cafeteria diet for 3 weeks. In spite of BAT activation, the diet promoted an increase in the carcass fatty acid content. Plasma insulin levels were markedly increased in cafeteria diet-fed rats. Two insulin-sensitive processes, in vivo fatty acid synthesis and in vivo glucose uptake (which was used to evaluate G3P generation via glycolysis) were increased in BAT from rats fed the cafeteria diet. Direct glycerol phosphorylation, evaluated by glycerokinase (GyK) activity and incorporation of [U-14C]glycerol into triacylglycerol (TAG)-glycerol, was also markedly increased in BAT from these rats. In contrast, the cafeteria diet induced a marked reduction of BAT glyceroneogenesis, evaluated by phosphoenolpyruvate carboxykinase-C activity and incorporation of [1-14C]pyruvate into TAG-glycerol. BAT denervation resulted in an approximately 50% reduction of GyK activity, but did not significantly affect BAT in vivo fatty acid synthesis, in vivo glucose uptake, or glyceroneogenesis. The data suggest that the supply of G3P for BAT TAG synthesis can be adjusted independently from the sympathetic nervous system and solely by reciprocal changes in the generation of G3P via glycolysis and via glyceroneogenesis, with no participation of direct phosphorylation of glycerol by GyK.

Glyceroneogenesis and the supply of glycerol-3-phosphate for glyceride-glycerol synthesis in liver slices of fasted and diabetic rats.

The pathways of glycerol-3-phosphate (G3P) generation for glyceride synthesis were examined in precision-cut liver slices of fasted and diabetic rats. The incorporation of 5 mM [U-(14)C]glucose into glyceride-glycerol, used to evaluate G3P generation via glycolysis, was reduced by approximately 26-36% in liver slices of fasted and diabetic rats. The glycolytic flux was reduced by approximately 60% in both groups. The incorporation of 1.0 mM [2-(14)C]pyruvate into glyceride-glycerol (glyceroneogenesis) increased approximately 50% and approximately 36% in slices of fasted and diabetic rats, respectively, which also showed a two-fold increase in the activity phosphoenolpyruvate carboxykinase. The increased incorporation of 1.0 mM [2-(14)C]pyruvate into glyceride-glycerol by slices of fasted rats was not affected by the addition of 5 mM glucose to the incubation medium. The activity of glycerokinase and the incorporation of 1 mM [U-(14)C]glycerol into glyceride-glycerol, evaluators of G3P formation by direct glycerol phosphorylation, did not differ significantly from controls in slices of the two experimental groups. Rates of incorporation of 1 mM [2-(14)C]pyruvate and [U-(14)C]glycerol into glucose of incubation medium (gluconeogenesis) were approximately 140 and approximately 20% higher in fasted and diabetic slices than in control slices. It could be estimated that glyceroneogenesis by liver slices of fasted rats contributed with approximately 20% of G3P generated for glyceride-glycerol synthesis, the glycolytic pathway with approximately 5%, and direct phosphorylation of glycerol by glycerokinase with approximately 75%. Pyruvate contributed with 54% and glycerol with 46% of gluconeogenesis. The present data indicate that glyceroneogenesis has a significant participation in the generation of G3P needed for the increased glyceride-glycerol synthesis in liver during fasting and diabetes.

Glyceroneogenesis is reduced and glucose uptake is increased in adipose tissue from cafeteria diet-fed rats independently of tissue sympathetic innervation.

The pathways of glycerol-3-P (G3P) generation were examined in retroperitoneal (RETRO) and epididymal (EPI) adipose tissues from rats fed a cafeteria diet for 3 wk. The cafeteria diet induced marked increases in body fat mass and in the plasma levels of insulin and triacylglycerol (TAG). RETRO and EPI from cafeteria diet-fed rats had increased rates of norepinephrine turnover (143 and 60%, respectively) and of de novo fatty acid (FA) synthesis (58 and 98%), compared with controls fed a balanced commercial diet. Cafeteria diet feeding induced marked increases in RETRO and EPI in vivo rates of glucose uptake (52 and 51%, respectively), used to evaluate G3P generation via glycolysis, as well as in glycerokinase activity (119 and 36%) and TAG-glycerol synthesis from glycerol (56 and 71%, respectively). In contrast, there was a marked reduction of glyceroneogenesis in RETRO and EPI from cafeteria diet-fed rats, which was evidenced by the significant decreases of P-enolpyruvate carboxykinase (PEPCK-C) activity (48 and 36%) and TAG-glycerol synthesis from pyruvate (45 and 56%, respectively). Denervation of RETRO from cafeteria diet-fed rats reduced the activity of glycerokinase by 50%, but did not affect glucose uptake or PEPCK-C activity and TAG-glycerol synthesis from pyruvate by the tissue. The data show that glyceroneogenesis can also be inhibited to adjust the supply of G3P to the existing rates of FA esterification and TAG synthesis and suggest that this adjustment is made by reciprocal changes in the generation of G3P from glucose via glycolysis and from glyceroneogenesis, independently from G3P production by glycerokinase.